Panorama™ Human v1 Array

Technical Bulletin

Catalog Number HPFM3 Panorama™ Human Kinase v1 Array

Table of Contents

Introduction ...... 1 Panorama Array Technology ...... 1 Kit Contents ...... 1 Storage Conditions ...... 2 General Recommendations ...... 2 Kit Components ...... 3 Protein Arrays ...... 3 Anti-phosphotyrosine-Cy5...... 4 Anti-c-Myc-Cy3 ...... 4 Control Assays ...... 4 Protocol for Autophosphorylation Assay ...... 4 Protocol for the Anti-c-Myc-Cy3 Binding Assay ...... 6 Scanning ...... 7 Data Analysis ...... 7 Data Normalization ...... 8 Standard Normalization...... 8 Guidelines for Different Assay Types ...... 9 General Recommendations ...... 9 on Arrays Using Exogenous ...... 9 Assays Utilizing Radioactive ATP ...... 9 DNA Binding Assays ...... 10 Antibody Binding Assays ...... 10 Protein:Protein Interactions ...... 10 Post-translational Modifi cation ...... 10 Troubleshooting Guide ...... 11 Appendices ...... 12 Appendix A: Protein Array Orientation ...... 12 Appendix B: Panorama Human Protein Function Array Kinase v1—Protein Identities ...... 13 Control and Marker Proteins ...... 17 Dilution Series ...... 17 References ...... 19 Relevant Patents ...... 19 Introduction The Panorama™ Human Kinase v1 Array contains 152 kinase proteins and specifi c controls. Kinases are categorized into familes based on their structure and activity and are known to regulate the majority of cellular pathways, especially those involved in and transmission of signals within the cell. Loss of kinase function has been linked to many human diseases, such as cancer1, and is known to affect cascades of reversible phosphorylation. Kinases are attractive targets for therapeutic drugs because they have similar structures and highly conserved ATP binding sites. Drugs which inhibit specifi c kinase activity such as Gleevec® and Iressa® are currently in clinical use for the treatment of numerous malignancies. Panorama Array Technology The kinases on the Panorama Human Kinase v1 Array were expressed in Sf9 insect cells and affi nity purifi ed directly on the array via their biotin tag. As a result of the proprietary BCCP (biotin-carboxyl carrier protein) tagging technology, all kinases are presented in a similar orientation while providing a 50 Å spacer arm to maximize the opportunity for sites to interact with binding partners2,3,4. Open reading frames (ORFs) are cloned in frame with two tag sequences at the C terminus encoding the BCCP tag and the c-Myc epitope (EQKLISEEDL), which can be used to visualize and quantitate the proteins on the array. To ensure fi delity, clones are sequence-verifi ed immediately prior to expression in Sf9 insect cells. During expression, the BCCP tag is biotinylated only when it is correctly folded5. All expressed proteins are assayed for incorporation of biotin, and Western blot analysis is used to determine molecular weight, confi rm biotinylation, and establish full-length protein has been expressed. Biotinylation of BCCP occurs at a single surface-exposed lysine residue approximately 50 Å from the attachment to the fusion protein. The BCCP-biotin-fusion proteins are captured on the array surface via a streptavidin-biotin interaction with BCCP acting as a spacer between the array substrate and the fusion protein. BCCP-biotin provides a single-point high-affi nity anchor so that all proteins on the array are in the same orientation. As a result, the arrayed proteins are not sterically or functionally hindered by multiple non-specifi c interactions with the surface and are freely available to interact with biochemical probes presented in solution, thereby minimizing non-specifi c interactions6. Panorama functional protein arrays are fabricated on borosilicate glass slides that display high chemical resistance, low auto-fl uorescence, and excellent surface uniformity. The slides are cut by a laser to minimize particle contamination. The slides are then coated with streptavidin that is covalently attached to a permeable three-dimensional coating comprised of a cross-linked matrix with low non-specifi c protein-binding. The format is compatible with conventional microarray scanners and instrumentation. Kit Contents

Product Cat. No. Size Panorama Human Kinase v1 Microarrays P2374 2 each Anti-phosphotyrosine-Cy5 T3576 10 µL Anti-c-Myc-Cy3 C6594 10 µL

Functional Assay Buffer C0492 150 mL Assay Buffer A1105 150 mL

Bovine Serum Albumin (BSA) A3059 2 3 200 mg 1M Dithiothreitol (DTT) 646563 1 ampule

1 Product Cat. No. Size 100 mM ATP A6559 100 µL quadriPERM® culture vessel Z376760 2 each HybriSlip™ H0784 10 each Pap Jar P8123 4 each 50 mL Conical Centrifuge Tubes C8296 2 each Panorama Kinase Array v1 Analysis Workbook and K4139 1 each GAL fi le (CD) Materials Required But Not Provided • High-purity water • Powder-free gloves • Microarray scanner or fl uorescence imager • Microarray analysis software • Forceps (fi ne and blunt-ended) • Centrifuge • Shaking incubator • Orbital shaker • Lint free tissue paper (e.g., Kimwipes) Storage Conditions Proteins on the array are sensitive to heat and oxidation. To preserve protein activity, the arrays are shipped on dry ice in screw-capped Pap jars and fi lled with 30 mL of storage buffer containing dithiothreitol and glycerol. Upon receipt, store the kit at –20 °C until use. The storage buffer for the protein arrays may be frozen upon arrival due to the dry ice used for shipping, but will thaw gently when placed at –20 °C. Once the storage buffer has thawed, open Pap jars only prior to use. General Recommendations a. The array area covers most of the slide surface; therefore, extreme care is needed in handling the arrays. Remove the arrays from their storage buffer by the labeled end using blunt-ended forceps. Do not touch the unprotected portion of the slide surface. b. Keep arrays in ice-cold buffer unless higher temperatures are required for assays. c. Always keep the array label-side upwards when lying fl at. To remove components of the storage buffer, wash arrays as indicated in the protocols. d. Cover arrays completely in assay buffer/reagents to prevent them from drying out during the assay.

2 e. If using critical volume sample, use the HybriSlip provided with the kit. Do not use glass cover slips as they may sequester the sample. Pipette 50 µL of sample carefully onto the middle of the slide and lower the cover slip gently onto the surface using fi ne forceps. If sample is not limited and larger volume incubations are possible, perform incubations in plastic quadriPERM containers provided with the kit. Use suffi cient sample solution to immerse the arrays. Assays in 2–5 mL of probing solutions yield the best results. f. Protect fl uorescent probes such as Cy-dye-labeled ligands from light during the assay. Cover the array with aluminum foil. g. When compatible with assay conditions and detection methods, include 20% glycerol and 0.1% Triton X-100 in buffers. h. The kinase arrays have been pre-blocked with 2% BSA during production. Additional blocking with non-fat milk powder or other commonly used blocking agents may be necessary. i. If a high degree of background speckling is observed after processing, use despeckling algorithms that are present in most commercially available microarray analysis software. Please note that speckling affects the appearance of the slide image only and does not materially affect the quantitative data generated. Kit Components Protein Arrays Each Kinase v1 array contains 152 protein kinases, numerous control features, and 48 markers in 48 sub-grids per slide. The protein kinases within the arrays have been printed in quadruplicate in 5 × 4 subarrays. Each feature on the array has a spot diameter of approximately 500 µm spaced at intervals of 300 µm. The arrayed kinase proteins are listed in Appendix B. Every fusion protein is expressed from a full-length ORF and each clone is fully sequence verifi ed prior to protein expression. The sequence of the cloned ORFs and links to protein databases can be downloaded from the Sigma-Aldrich Web site (sigma-aldrich.com). A schematic and key to the Kinase v1 array are shown in Appendices A and B. Each Kinase v1 array contains: • 152 kinase proteins • 48 Cy3/Cy5-labeled BSA marker spots for array alignment • 4 negative control spots consisting of the immobilization tag, BCCP • 4 negative control spots consisting of b-galactosidase-BCCP • 4 anti-c-Myc positive control spots consisting of b-galactosidase-c-Myc • 4 anti-c-Myc positive control spots consisting of b-galactosidase-BCCP-Myc • Dilution series of 12 anti-c-Myc positive control spots consisting of b-galactosidase-BCCP-Myc • Dilution series of 12 phosphotyrosine peptide control spots consisting of oriented peptides joined to the surface via a linker and consisting of the sequence, biotin-Ahx-KVEKIGEGT[pY]GVVKK- CONH2, in which the tyrosine residue is phosphorylated. • Dilution series of 12 kinase substrate control spots consisting of oriented peptides joined to the surface via a linker and consisting of the sequence biotin-Ahx-KVEKIGEGTYGVVKK-CONH2, which acts as a substrate for certain tyrosine kinases.

3 • Dilution series of negative control spots consisting of biotin-BSA • 36 sets of negative control spots printed with lysis buffer The proteins are arrayed onto a streptavidin-coated, low fl uorescence glass slide (25 × 75.6 × 1 mm) that is compatible with conventional microarray instrumentation. The upper side is indicated by the label (Appendix A). The arrays are pre-washed and blocked with 2% BSA to reduce non-specifi c binding. If additional blocking is desired, see Assay Guidelines for recommendations. Anti-phosphotyrosine-Cy5 To validate the functionality of proteins on the Kinase v1 array, an autophosphorylation assay using Cy5-labeled anti-phosphotyrosine is supplied as a control. In the control assay, ATP is applied to the slide and phosphorylation of tyrosine is detected by the labeled monoclonal antibody. Although some proteins become phosphorylated when they are expressed in Sf9 cells, incubation of the array in buffer containing ATP leads to higher levels of phosphorylation of particular proteins (Figure 1). Autophosphorylation and detection should be carried out using the buffers provided with this kit. Anti-c-Myc-Cy3 A Cy3-labeled anti-c-Myc monoclonal antibody is provided and serves as a positive control because the c-Myc tag is incorporated into all protein kinases on the array. It is recommended to use the anti-c-Myc to probe each control or experimental array immediately after performing primary assays for protein function. This assay demonstrates that proteins are immobilized at each spot and can be used to quantify the amount of protein for data normalization (Figure 2). Perform the binding assay with the Assay Buffer provided with the kit. Control Assays Protocol for Autophosphorylation Assay a. Allow the Functional Assay Buffer to equilibrate to room temperature. In order to perform the assay, it is necessary to make 2 buffers from the Functional Assay Buffer. b. Prior to performing the Autophosphorylation Assay, add 150 µL of 1 M DTT to the bottle containing 150 mL of Functional Assay Buffer and place at room temperature. c. Prepare the Anti-phosphotyrosine Probing Buffer: add 200 mg of BSA to 10 mL of Functional Assay Buffer+DTT from step b. Mix gently to dissolve. d. Prepare the Phosphorylation Assay Buffer+ATP: add 10 µL of 100 mM ATP solution to 10 mL of Functional Assay Buffer+DTT from step b. Mix gently by inversion. e. Remove array from storage buffer and place in a clean Pap jar containing 25 mL of Functional Assay Buffer+DTT. Place the Pap jar on ice and shake gently (40–50 rpm) for 5 min. f. Remove the array from the Functional Assay Buffer+DTT and drain the slide by blotting the long edge carefully onto lint-free tissue paper for 10 seconds. g. Dry the back of the slide with lint-free tissue paper.

4 h. Place the slide into a chamber of a quadriPERM culture dish so that it is horizontal with array side up. Ensure the slide does not rest on the plastic lugs at the numbered end of the chamber as this may lead to incomplete coverage of the slide. Immediately pipette 5 mL of the Phosphorylation Assay Buffer+ATP carefully onto the slide, while minimizing introduction of bubbles. Add any additional slides to the chambers in the same manner, then replace the lid. i. Place the chamber in a shaking incubator at 30 ºC. Ensure the assay solution covers the entire array. Shake the dish gently for 30 min at 30 ºC. j. During the incubation, dilute the Anti-phosphotyrosine-Cy5 conjugate 1:1000 (6 µL plus 6 mL of Anti-phosphotyrosine Probing Buffer). k. After incubation, carefully remove each array from the incubation dish with a pair of forceps and place in a clean Pap jar containing 25 mL of Functional Assay Buffer+DTT. Cap the tube, then invert several times. Place the tube on a shaker and shake gently at room temperature for 5 min to remove unbound probe. Pour off the Functional Assay Buffer+DTT. l. Repeat the wash once more, inverting the container several times at each wash step. m. Remove the array from the Functional Assay Buffer+DTT and drain the slide by blotting the long edge carefully onto lint-free tissue paper for 10 seconds. n. Dry the back of the slide with lint-free tissue paper. o. Place the slide into a chamber of a quadriPERM culture dish, as before. Ensure the slide does not rest on the plastic lugs at the numbered end of the chamber as this may lead to incomplete coverage of the slide. Immediately pipette 5 mL of the diluted Anti-phosphotyrosine-Cy5 conjugate from step j onto the slide, while minimizing introduction of bubbles. Add any additional slides to the chambers in the same way and replace the lid. p. Place the chamber on an orbital shaker at room temperature and ensure the assay solution covers the entire array. Cover with foil to protect from light and shake dish gently for 30 min at room temperature. q. After incubation, carefully remove each array from the incubation chamber with a pair of forceps and place in a clean Pap jar containing 25 mL of Functional Assay Buffer+DTT. Cap the tube, then invert several times. Shake gently for 5 min to remove unbound probe. Pour off the Functional Assay Buffer+DTT. r. Perform two more 5 min washes, inverting the container several times at each wash step. s. Pour off the fi nal Functional Assay Buffer-DTT and add 25 mL of high purity water to wash away glycerol. Replace the lid on the container and invert several times before pouring off the water. t. Immediately transfer the arrays to a 50 mL disposable centrifuge tube with the slide label at the bottom of the tube, using extreme care only to touch the slide label or edges to prevent damage to the array. Centrifuge the arrays at room temperature for 2 min at 240 3 g. u. Carefully remove the slides from the centrifuge tubes with a pair of dry, blunt-ended forceps, touching only the extreme end of the slide. v. Scan the slides from the non-labeled end using a microarray scanner or imager.

5 Protocol for the Anti-c-Myc-Cy3 Binding Assay After performing the functional assay, use the anti-c-Myc-Cy3 conjugate to demonstrate the level of protein present in each kinase protein feature. a. Allow the Assay Buffer to equilibrate to room temperature. Perform all steps at room temperature. In order to perform the assay, it is necessary to make two buffers from the Assay Buffer. b. Prepare the Anti-c-Myc Wash Buffer+DTT: add 150 µL of 1 M DTT to 150 mL of Assay Buffer immediately before use. c. Prepare the Anti-c-Myc Probing Buffer: add 200 mg of BSA to 10 mL of Anti-c-Myc Wash Buffer+DTT from step b. d. Dilute the anti-c-Myc-Cy3 conjugate1:1000 (5 µL plus 5 mL of anti-c-Myc Probing Buffer+DTT.) e. Place slides in a clean Pap jar with 25 mL of Anti-c-Myc Wash Buffer+DTT and incubate with gentle shaking (40–50 rpm) for 5 min. f. Remove each array with a pair of blunt-ended forceps. Drain excess liquid from the slide surface by resting the long edge of the slide on lint-free tissue paper for 10 seconds. g. Dry the back of the slide on lint-free tissue paper. h. Place the slide into a chamber of a quadriPERM culture dish so it is level horizontally with the array side facing up, then immediately pipette 2 mL of the diluted anti-c-Myc-Cy3 carefully onto the slide without introduction of bubbles. Add additional slides to the chambers in the same manner, then replace the lid. Ensure the slide does not rest on the plastic lugs at the numbered end of the chamber as this may lead to incomplete coverage of the slide. To protect the fl uorophore, cover the quadriPERM vessel with foil. i. Incubate the quadriPERM vessel at room temperature and shake gently for 6 hours. Most c-Myc positive proteins on the array can be visualized after a 6-hour incubation. To increase assay sensitivity, the incubation can be extended overnight up to 20 hours before processing. For longer incubations, place the quadriPERM dish in a sealed plastic container with a wad of damp tissues to prevent drying out. j. After incubation, carefully remove each array from the incubation chamber with a pair of forceps and place in a clean Pap jar with 25 mL of Anti-c-Myc Wash Buffer+DTT (two arrays per tube). Cap the tube, then invert several times. Shake the tube gently for 5 min to remove unbound antibody. Pour off the Anti-c-Myc Wash Buffer+DTT. k. Repeat two more times, inverting the container several times at each wash step. l. Pour off the fi nal Anti-c-Myc Wash Buffer+DTT and add 25 mL of water to the Pap jar. Cap the tube and invert several times to remove glycerol from the slide surface. m. Using forceps, place each slide in a 50 mL disposable centrifuge tube with the label at the bottom of the tube. Be careful only to handle slides by the label or by the edges of the slide to avoid damaging the array. Centrifuge the arrays at room temperature for 2 min at 240 × g. n. Carefully remove the slides from the centrifuge tubes with a pair of dry forceps, touching only the extreme end of the slide. o. Scan the slides from the non-labeled end fi rst using a microarray scanner or imager.

6 Scanning a. Protein function arrays can be scanned using conventional microarray scanners and imagers to detect fl uorescence. b. Scanning should be performed according to the manufacturer’s recommended protocols. Care should be taken not to scratch the array surface during handling. c. Excitation and emission wavelengths for Cy5 are lex 649 nm and lem 670 nm, respectively. Cy3 excitation and emission are lex 550 nm and lem 570 nm, respectively. d. It is advisable to perform a pre-scan to adjust the scanning parameters and obtain an optimal image. Final images should have the following properties: the most intense spots should not be saturated and background should be as low as possible. If the array image is faint, repeat the scanning with increased Laser Power (%) and/or gain (PMT voltage) as necessary. In the opposite case, repeat the scanning process at lower Laser Power (%) and/or lower PMT settings. Scanning should be repeated to obtain an optimal image, bearing in mind that “overexposure” can lead to photobleaching of fl uorophores. e. Images should be saved as TIFF fi les for future analysis. Data Analysis Analyze the images using a standard microarray analysis software package. After loading the TIFF fi le, analyze the array with an array-specifi c grid using the GAL fi le provided with the kit. The GAL fi le will also provide annotation for each spot on the array. Once the GAL fi le is loaded, adjust the grid so the marker spots are aligned in the top left corner of each sub-grid. This may be followed by auto-alignment. After auto-alignment, it may be necessary to manually edit individual subarrays to ensure optimal alignment. Follow the guidelines of the software manufacturer. For optimal results, use local background subtraction and measure net signal intensities using median pixel intensity values. Set the spot size to 400 µm. When the grid alignment is complete, export the data in the order determined by the GAL fi le to the Excel spreadsheet provided with the kit. Cut and paste the column of data for each slide into the blue columns in the Cy3 or Cy5 “Input” sheets of the Panorama Kinase Array v1 Analysis Workbook. The Excel spreadsheet automatically sorts the data and determines the mean and standard deviation of the four replicates for each protein feature on the array. If required, sort the processed data manually according to the spot intensity and display the data graphically using standard Excel procedures.

7 Data Normalization The amount of material for each protein on the array can vary depending on expression levels. Take the variation of protein amount into consideration when analyzing data from microarray experiments. The Kinase v1 array includes biotin-BSA to detect any general non-specifi c binding. Other controls include proteins with and without the c-Myc epitope tag (b-galactosidase-BCCP-Myc, b-galactosidase-BCCP, BCCP-Myc and BCCP), which will indicate any non-specifi c interactions related to the folding and detection tags. The controls measure any non-specifi c interactions and set a background level against which to determine signifi cant interactions. Perform the anti-c-Myc-Cy3 assay on each slide after the primary assay to measure the amount of protein in every spot. The data obtained from the anti-c-Myc-Cy3 assay can be used to normalize the data from other assays performed on the array. Standard Normalization The signal from the primary assay is divided by the signal from the anti-c-Myc-Cy3 assay, thereby allowing normalization of the assay result to the amount of protein in each spot. After determining the mean and standard deviation for each of the four replicates for each protein, plot the data. Note: If values for the anti-c-Myc-Cy3 conjugate assay are particularly low, do not use the values for normalization as this may lead to unreliable normalization of the primary assay data. As a guide, do not normalize values in the anti-c-Myc-Cy3 conjugate assay that are not signifi cantly greater than the binding to control proteins using this method. Detection of individual proteins using the anti-c-Myc-Cy3 assay can be affected by the scanning parameters used, prior functional assays on the array, and occlusion of the c-Myc epitope. As with any kind of normalization, consider whether normalization has introduced any misleading results. The following criteria should be used to corroborate positive results: • Check magnitude of non-normalized data in both assays to ensure the normalized data is realistic. • Ensure data for each of the four replicate spots display the same trend. • Visually check positives on the original array images. As with any array technology, validate any positive data spots by independent techniques such as immunoprecipitation assays or electrophoretic mobility shift.

8 Guidelines for Different Assay Types General Recommendations Design buffers in accordance with known literature for the particular assay under investigation, taking due consideration of the need for particular ions or cofactors. Where suitable, it is recommended that all buffers include 20% glycerol, 1 mM DTT, 0.1% Triton X-100, and at least 0.1% BSA to stabilize proteins on the array. Kinase v1 arrays have been blocked with 2% BSA; however, depending on the assay, it may be necessary to block further. To minimize non-specifi c binding, perform protein interaction and antibody binding assays in the presence of blocking materials such as 2% BSA or dried low-fat milk powder. Blocking with 5% milk powder is not recommended for work involving phosphorylation as it contains high concentrations of phosphoproteins that can increase background. If biotinylated probes are to be used, block the streptavidin surface with buffers including 20 µM biotin. In such cases, it is advisable to probe in buffers containing 5% dried low-fat milk powder. When labeling proteins or peptides with fl uorescent dyes, do not exceed a labeling ratio of 1–2 dye moieties per molecule to ensure there is minimal interference with binding. Label peptide and oligonucleotide probes during synthesis and purify them via HPLC. An aminohexanoic acid spacer between the fl uorophore and peptide is appropriate for short sequences in which steric hindrance may be problematic. Store labeled probes (e.g., DNA, peptides, proteins, or antibodies) in aliquots at –20 ºC. Do not freeze/ thaw aliquots more than once. Where applicable, perform assays at 4 ºC to maximize protein stability. Phosphorylation on Arrays Using Exogenous Kinases To determine which proteins are potential substrates for particular kinases, the proteins on the Kinase v1 array can be used as substrates for exogenous kinases. Use the protocol for the Autophosphorylation Assay described in this guide as the basis for development of assays using exogenous kinases. Refer to General Recommendations for additional information. Phosphorylation events can be successfully detected using labeled anti-phosphotyrosine, anti- phosphoserine, anti-phosphothreonine, or radiolabelled ATP. Add exogenous kinases to the probing solution at a concentration in the region of 10 nM. Perform phosphorylation reactions at 20–30 ºC. Assays Utilizing Radioactive ATP Phosphorylation of kinases on the Kinase v1 array can be detected using radiolabeled ATP. To perform phosphorylation using radiolabeled ATP, follow the general procedure described for the Autophosphorylation Assay. Ensure the fi nal concentration of ATP exceeds 100 µM and has a specifi c activity of >60 Ci/mmol [g-33P]-ATP. Exogenous kinases can be added to the reaction at a concentration in the range of 10 nm. To reduce the amount of radioactivity used, perform incubations in low volumes. Perform assays using 50 µL of probing solution under a HybriSlip supplied with this kit. Exogenous kinases may autophosphorylate in the presence of ATP. It is important to ensure the kinase is thoroughly removed from the array before detection. To reduce non-specifi c binding of the exogenous

9 kinase or radiolabeled probe to the array surface, wash the arrays once with water, followed by two washes with 0.5% SDS in water, then wash the arrays twice in high purity water to remove the detergent before drying and detection. Perform each wash for 5 min with shaking at room temperature. Phosphorylation can be imaged using autoradiography or phosphorimaging. DNA Binding Assays It is recommended that oligonucleotides are 5’-labeled with fl uorophores during synthesis (available from Sigma-Genosys). Both strands of double-stranded DNA probes can be labeled to maximize sensitivity. When selecting probes, it is recommended to apply the same considerations as for any other DNA: protein study. Probes should be of the highest purity possible to generate unequivocal results. Oligonucleotides should not include repeat or lengthy sequences that may give rise to secondary structures. Store aliquots of oligonucleotides at –20 °C. Do not freeze/thaw aliquots of probe more than once. Antibody Binding Assays Antibody labeling may be performed using mono-reactive Cy-dyes. Desalt labeled probes to remove free dye before use. Some dye labels lead to higher levels of non-specifi c binding than others. If high non-specifi c binding is observed, it may be necessary to evaluate other dyes. When preparing labeled antibodies, prepare aliquots and store at –20 °C. Do not freeze/thaw aliquots of antibodies more than once. Antibody binding can be detected by direct labelling of the antibody or by secondary detection using another antibody or probe. To simplify assay development, it is recommended to use direct detection where applicable. Protein:Protein Interactions The optimal conditions for studying protein:protein interactions on the array will vary according to the protein being studied. The optimal concentration of protein probe will depend on the affi nity of interaction with arrayed proteins. Choose the probe concentration in accordance with the known literature or experience. Protein:protein interactions can be detected by direct labeling or by indirect detection using labeled antibodies or probes. To simplify assay development, it is recommended to use direct detection where applicable. Post-translational Modifi cation Protein function arrays can be used to determine whether proteins on the array are potential substrates for specifi c such as kinases. These arrays can be used to determine the effect of post- translational modifi cation in vitro on protein function. The proteins on the array can be modifi ed enzymatically on the array and detected using labeled antibodies specifi c for a particular modifi cation.

10 Troubleshooting Guide Signal is very low over the entire array • Fluorescent labeling of the molecule used as probe may not have been effi cient. Check the degree of labeling of the probe (1–2 molecules of dye per protein) and either repeat the labeling or use a higher amount of probe. • Assay conditions used may be sub-optimal for any new interaction or reaction under study. Optimize the conditions as appropriate including blocking conditions, buffer composition, assay incubation time, and temperature. • Check literature for specifi c conditions required for interaction of the probe with particular protein targets. Signal for replicate spots is inconsistent • When using cover slips, ensure that no air bubbles are trapped during assay. • When assaying in larger volumes, ensure the arrays are completely covered with liquid and not allowed to dry out during assay. • When using quadriPERM dishes, ensure the slide does not rest on the plastic lugs at the numbered end of the chamber. Streaking is observed on the array after scanning • This may occur when slides are dried by centrifugation and the array label is placed at the top. Always dry the slides with the label at the bottom of the tube. Spots on the array appear scratched • If using cover slips, be careful not to drag the cover slip across the array surface when placing or removing. • When using forceps, be certain to grasp the slide at either end and be certain not to touch the area where proteins are printed. The background is speckled • Use clean powder-free gloves when handling the arrays. • Ensure assay containers are clean and free of fl uorescent contaminants. • Where applicable, ensure buffers are prepared freshly before use. • Apply despeckling algorithms present in most commercially available microarray analysis software packages. Note: The speckling phenomenon does not materially affect the data generated. The background signal is high over the entire surface • Re-scan at lower laser power and/or PMT setting. • Further blocking before and/or during the assay may be required to prevent non-specifi c interactions. Blocking is assay and sample dependant. Try using commonly used blocking agents (i.e., non-fat milk or increased concentrations of BSA). • When using a labeled antibody probe, the concentration of the probe must be optimized to minimize non-specifi c binding. There are localized patches of high background on the slides • Ensure arrays are completely covered with buffer during wash steps. • Ensure arrays are not allowed to dry out during assay or processing.

11 Appendices Appendix A: Protein Array Orientation

The Human Kinase Array consists of forty-eight, 5 3 4 subgrids. Two Cy3/Cy5 biotin BSA marker spots are situated in the top left corner of the fi rst and fourth sub-arrays of each row of subpanels. A dilution series of control proteins is situated in the top line of the second column of sub-arrays.

Panorama™ Human Kinase Protein Array HPFM3

12 Appendix B: Panorama Human Protein Function Array Kinase v1—Protein Identities Entrez Symbol Name Gene ID AAK1 AP2 associated kinase 1 22848 ACVR1 activin A receptor, type I 90 ACVR1C activin A receptor, type IC 130399 ADCK4 aarF domain containing kinase 4 79934 ADRBK2 adrenergic, beta, receptor kinase 2 157 AKT1 v-akt murine thymoma viral oncogene homolog 1 207 ALS2CR2 amyotrophic lateral sclerosis 2 (juvenile) region, candidate 2 55437 APEG1 aortic preferentially expressed protein 1 10290 ARAF1 v-raf murine sarcoma 3611 viral oncogene homolog 1 369 AURKB serine/threonine kinase 12 9212 BLK B lymphoid tyrosine kinase 640 BMX BMX non-receptor tyrosine kinase 660 BRD3 bromodomain containing protein 3 8019 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) 699 BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) 701 C20orf64 / chromosome 20 open reading frame 64 112858 PRPK C20orf97 chromosome 20 open reading frame 97 57761 CAMK1G calcium/calmodulin-dependent IG 57172 CAMK2B calcium/calmodulin-dependent protein kinase (CaM kinase) II beta 816 CAMK2D calcium/calmodulin-dependent protein kinase (CaM kinase) II delta 817 CAMK2G calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma 818 CAMK4 calcium/calmodulin-dependent protein kinase IV 814 CAMKK1 calcium/calmodulin-dependent protein kinase kinase 1, alpha 84254 CAMKK2 calcium/calmodulin-dependent protein kinase kinase 2, beta 10645 CCRK cell cycle related kinase 23552 CDC2 cell division cycle 2, G1 to S and G2 to M 983 CDK2 cyclin-dependent kinase 2 1017 CDK4 cyclin-dependent kinase 4 1019 CDK5 cyclin-dependent kinase 5 1020 CDK7 cyclin-dependent kinase 7 1022 CDK9 cyclin-dependent kinase 9 1025 CDKN2A cyclin-dependent kinase inhibitor 2A 1029 CHEK1 CHK1 checkpoint homolog (S. pombe) 1111

13 Entrez Symbol Gene Name Gene ID CHEK2 CHK2 checkpoint homolog (S. pombe) 11200 CLK1 CDC-like kinase 1 1195 CLK2 CDC-like kinase 2 1196 CLK3 CDC-like kinase 3 1198 COL4A3BP Similar to collagen, type IV, alpha 3 (Goodpasture antigen) binding protein 10087 CSNK1D , delta 1453 CSNK1G1 casein kinase 1, gamma 1 53944 CSNK1G2 casein kinase 1, gamma 2 1455 CSNK2A1 , alpha 1 polypeptide 1457 CSNK2A2 casein kinase 2, alpha prime polypeptide 1459 DYRK2 dual-specifi city tyrosine-(Y)-phosphorylation regulated kinase 2 8445 DYRK4 dual-specifi city tyrosine-(Y)-phosphorylation regulated kinase 4 8798 FASTK FAST kinase 10922 FES feline sarcoma oncogene 2242 FGFR2 fi broblast growth factor receptor 2 2263 FLJ14813 hypothetical protein FLJ14813 84930 FLJ20574 hypothetical protein FLJ20574 54986 FRK fyn-related kinase 2444 FYN FYN oncogene related to SRC, FGR, YES 2534 GPRK6 G protein-coupled receptor kinase 6 2870 GSK3B glycogen synthase kinase 3 beta 2932 H11 protein kinase H11 26353 HK1 hexokinase 1 3098 IKBKB inhibitor of kappa light polypeptide gene enhancer in B-cells kinase beta 3551 ILK integrin-linked kinase 3611 JIK STE20-like kinase 51347 KIS kinase interacting with leukemia-associated gene (stathmin) 127933 LCK lymphocyte-specifi c protein tyrosine kinase 3932 LIMK2 LIM domain kinase 2 3985 MAK male germ cell-associated kinase 4117 MAP2K3 -activated protein kinase kinase 3 5606 MAP2K5 mitogen-activated protein kinase kinase 5 5607 MAP2K6 mitogen-activated protein kinase kinase 6 5608 MAP2K7 mitogen-activated protein kinase kinase 7 5609 MAP3K14 mitogen-activated protein kinase kinase kinase 14 9020 MAP3K6 mitogen-activated protein kinase kinase kinase 6 9064 MAP3K7 mitogen-activated protein kinase kinase kinase 7 6885 MAP4K5 mitogen-activated protein kinase kinase kinase kinase 5 11183

14 Entrez Symbol Gene Name Gene ID MAPK1 mitogen-activated protein kinase 1 5594 MAPK11 mitogen-activated protein kinase 11 5600 MAPK13 mitogen-activated protein kinase 13 5603 MAPK14 mitogen-activated protein kinase 14 1432 MAPK3 mitogen-activated protein kinase 3 5595 MAPK6 mitogen-activated protein kinase 6 5597 MAPK7 mitogen-activated protein kinase 7 5598 MAPK9 mitogen-activated protein kinase 9 5601 MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 7867 MARK3 MAP/microtubule affi nity-regulating kinase 3 4140 MATK megakaryocyte-associated tyrosine kinase 4145 MGC16169 hypothetical protein MGC16169 93627 MGC42105 hypothetical protein MGC42105 167359 MGC45428 hypothetical protein MGC45428 166614 MKNK1 MAP kinase-interacting serine/threonine kinase 1 8569 NEK11 NIMA (never in mitosis gene a)- related kinase 11 79858 NEK3 NIMA (never in mitosis gene a)-related kinase 3 4752 PACE-1 ezrin-binding partner PACE-1 57147 PAK4 p21(CDKN1A)-activated kinase 4 10298 PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) 5106 PCTK1 PCTAIRE protein kinase 1, transcript variant 2 5127 PCTK1 PCTAIRE protein kinase 1, transcript variant 3 5127 PCTK2 PCTAIRE protein kinase 2 5128 PDK1 pyruvate dehydrogenase kinase, isoenzyme 1 5163 PDK2 pyruvate dehydrogenase kinase, isoenzyme 2 5164 PDK3 pyruvate dehydrogenase kinase, isoenzyme 3 5165 PDK4 pyruvate dehydrogenase kinase, isoenzyme 4 5166 PHKG2 , gamma 2 (testis) 5261 PIK3C3 phosphoinositide-3-kinase, class 3 5289 PIK3R1 phosphoinositide-3-kinase, regulatory subunit, polypeptide 1 (p85 alpha) 5295 PIM1 pim-1 oncogene 5292 PIM2 pim-2 oncogene 11040 PKE PKE protein kinase 282974 PKLR pyruvate kinase, liver and RBC 5313 PKM2 pyruvate kinase, muscle 5315 PLK polo (Drosophia)-like kinase 5347

15 Entrez Symbol Gene Name Gene ID PRKACB protein kinase, cAMP-dependent, catalytic, beta 5567 PRKACG protein kinase, cAMP-dependent, catalytic, gamma 5568 PRKCB1 , beta 1 5579 PRKCH protein kinase C, eta 5583 PRKCI protein kinase C, iota 5584 PRKCN protein kinase C, nu 23683 PRKCZ protein kinase C, zeta 5590 PRKD2 protein kinase D2 25865 PTK2 PTK2 protein tyrosine kinase 2 5747 PTK9 PTK9 protein tyrosine kinase 9 5756 PXK PX domain containing serine/threonine kinase 54899 RAF1 v-raf-1 murine leukemia viral oncogene homolog 1 5894 RIOK2 RIO kinase 2 (yeast) 55781 RIOK3 RIO kinase 3 (yeast) 8780 RIPK2 receptor-interacting serine-threonine kinase 2 8767 RPS6KA1 ribosomal protein S6 kinase, 90kDa, polypeptide 1 6195 RPS6KA2 ribosomal protein S6 kinase, 90kDa, polypeptide 2 6196 RPS6KA5 ribosomal protein S6 kinase, 90kDa, polypeptide 5 9252 RPS6KL1 ribosomal protein S6 kinase-like 1 83694 SGK serum/glucocorticoid regulated kinase 6446 SGKL serum/glucocorticoid regulated kinase-like 23678 SNARK likely ortholog of rat SNF1/AMP-activated protein kinase 81788 SNK serum-inducible kinase 10769 SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) 6714 SRPK1 SFRS protein kinase 1 6732 STK11 serine/threonine kinase 11 (Peutz-Jeghers syndrome) 6794 STK16 serine/threonine kinase 16 8576 STK17B serine/threonine kinase 17b (-inducing) 9262 STK22C serine/threonine kinase 22C (spermiogenesis associated) 81629 STK22D serine/threonine kinase 22D (spermiogenesis associated) 83942 STK24 serine/threonine kinase 24 (STE20 homolog, yeast) 8428 STK25 serine/threonine kinase 25 (STE20 homolog, yeast) 10494 STK3 serine/threonine kinase 3 (STE20 homolog, yeast) 6788 STK33 serine/threonine kinase 33 65975 STK38 serine/threonine kinase 38 11329 STK38L serine/threonine kinase 38 like 23012 STK6 serine/threonine kinase 6 6790

16 Entrez Symbol Gene Name Gene ID SYK spleen tyrosine kinase 6850 TBK1 TANK-binding kinase 1 29110 TEK TEK tyrosine kinase, endothelial 7010 TLK1 tousled-like kinase 1 9874 TTK TTK protein kinase 7272 VRK3 vaccinia related kinase 3 51231 ZAK sterile alpha motif and containing kinase AZK 51776 ZAP70 zeta-chain (TCR) associated protein kinase 70kDa 7535

Control and Marker Proteins

Protein Gene Content Code Symbol Identify ID Marker Cy3/Cy5 labeled biotin-BSA marker proteins

Control 1 b-gal-BCCP-Myc Control 2 BCCP-Myc

Control 3 b-gal-BCCP Control 4 BCCP Controls 5–12 Insect Lysis Buffer

Dilution Series Features BCCP1 to BCCP6 represent a two-fold dilution series of BCCP-Myc starting from the standard lysate concentration used for all other recombinant proteins. Features BSA1 to BSA 6 represent a two-fold dilution series of biotin-BSA starting at a spotting concentration of 25 ng/µL.

Biotin BSA Standard Spotting Concentration BSA1 25 ng/µL BSA2 12.5 ng/µL BSA3 6.25 ng/µL BSA4 3.125 ng/µL BSA5 1.56 ng/µL BSA6 0.78 ng/µL

17 Features NP1 to NP6 represent a two-fold dilution series of a non-phosphorylated peptide (biotin-Ahx- KVEKIGEGTYGVVKK-CONH2) which acts as a substrate for certain kinases such as FES kinase.

Non-phosphorylated Peptide Spotting Concentration Standard NP1 156 pg/µL

NP2 78 pg/µL NP3 39 pg/µL NP4 19.5 pg/µL NP5 9.8 pg/µL NP6 4.9 pg/µL

Features PP1 to PP6 represent a two-fold dilution series of a phosphorylated peptide (biotin-Ahx- KVEKIGEGT[pY]GVVKK-CONH2) which is recognized by anti-phosphotyrosine Cy5 supplied with this kit.

Phosphorylated Peptide Spotting Concentration Standard PP1 156 pg/µL PP2 78 pg/µL PP3 39 pg/µL PP4 19.5 pg/µL PP5 9.8 pg/µL PP6 4.9 pg/µL

18 References 1. A census of human cancer (2004) Nature Reviews Cancer, 4, 177-183, Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, Rahman N and Stratton MR. 2. Functional protein microarrays for parallel characterisation of p53 mutants (2004) Proteomics, 4, 1950-1958, Boutell JM, Hart DJ, Godber BLJG, Kozlowski RZ and Blackburn JM. 3. Strategies for immobilisation of biomolecules in a microarray (2004), Combinatorial Chemistry & High Throughput Screening, 7, 213-221, Yeo DSY, Panicker RC, Tan L-P and Yea SQ. 4. Enzymatic activity on a chip: The critical role of protein orientation (2005) Proteomics, 5, 416-419, Cha TW, Guo A and Zhu X-Y. 5. Development of Protein Microarrays for Drug Discovery (2004) Koopmann JO, McAndrew MB and Blackburn JM in Protein Microarrays; Mark Schena (Ed), pp 401-420. 6. Reversible, site specifi c immobilization of polyarginine-tagged fusion proteins on mica surfaces. (1997) FEBS Letters 414, 233-238, Nock S., Spudich, J.A., Wagner, P. Relevant Patents 1. WO 01/57198. Methods Of Generating Protein Expression Arrays And The Use Thereof In Rapid Screening. 2. WO 02/27327. Rapid Profi ling Of The Interactions Between A Chemical Entity And Proteins In A Given Proteome. 3. WO 03/048768. Protein Arrays For Allelic Variants And Uses Thereof. 4. WO 03/064656. Protein Tag Comprising A Biotinylation Domain and Method for Increasing Solubility and Determining Folding State.

19 Notes

20 Notes

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